The objective of this application is to examine the contributions of the EGF receptor family members ErbB1 and ErbB2/ErbB3 in vivo motility, tumor cell intravasation and metastasis. A better understanding of how tumor cells enter blood vessels will enhance our ability to develop therapies and diagnostic tests for improving treatment of patients with cancer. We have found in the previous cycle that increased ErbB1 or ErbB2/ErbB3 signaling can enhance tumor cell intravasation and metastasis, without affecting in vivo growth rates. We hypothesize that the increased chemotactic responses that we observe in vitro and increased invasive responses observed in vivo enable metastatic tumor cells to detect leaky vessels and move towards them, resulting in enhanced intravasation. In Specific Aim 1, we perform an analysis of the C terminal tyrosines on ErbB2 and ErbB3 that are required for enhancement of intravasation and metastasis. In Specific Aim 2, we use multiphoton microscopy imaging to evaluate tumor cell migration in the primary tumor relative to blood vessels and test the roles of ErbBs and vessel leakiness in directly stimulating tumor cell motility. In Specific Aim 3, we test whether external gradients of ligand are required for enhancement of tumor cell motility, intravasation and metastasis. This proposal makes use of orthotopic, in vivo models of spontaneous metastasis together with intravital imaging of primary tumors to perform unique measurements of the contributions of ErbBs on intravasation and metastasis.